Poster abstracts

Poster number 14 submitted by Aparna Unnikrishnan

Protein Dynamics Underlie Cre-loxP DNA Recombination

Aparna Unnikrishnan (Biophysics Graduate Program), Carlos D. Amero (CIQ, Universidad Autnoma del Estado de Morelos, Mexico), Mark P. Foster (Department of Chemistry and Biochemistry, OSU)

Abstract:
Cre recombinase (38.5 kDa, 343 residues), belonging to the tyrosine recombinase family, mediates site-specific recombination between specific loxP DNA recognition sequences. Two Cre molecules bind to each 34 bp loxP sequence and further associate to form an antiparallel tetrameric synaptic complex. Crystal structures show that the DNA duplexes bend ~80o upon formation of the synaptic tetrad, and although crystallographic, single molecule, and biochemical data implicate protein conformational changes (i.e., dynamics) in the progression of the Cre-loxP recombination pathway, the motions, timescales, amplitudes, and even their structural basis remain poorly understood. We aim to characterize the dynamics of Cre recombinase at atomic resolution using solution NMR spectroscopy through measurements of backbone amide and side chain methyl relaxation rates. 15N T1, T2 and {1H}-15N heteronuclear NOE relaxation measurements on the free catalytic domain of Cre identified several regions that exhibit fast dynamics on the ps-ns timescales. Two of these flexible regions, the β2-β3 loop and helix αN, form part of the inter-protomer interface in the synaptic tetramer, while the αJ-αK loop contacts the DNA phosphate backbone. These findings provide insight into the solution structure of Cre and are consistent with the hypothesis that protein dynamics are important for mediating DNA recognition and synaptic assembly. Measurement of dynamics spanning a range of timescales will be performed to characterize the motions of Cre in the absence and presence of DNA. While the role played by induced fit in Cre function has long been inferred, NMR methods tailored to large systems represent a route to unprecedented clarity in the mechanism of DNA substrate recognition and site specific recombination.

References:
1. Van Duyne, G. D. A Structural View of Cre-loxP Site-Specific Recombination. Annu. Rev. Biophys. Biomol. Struct. 30, 87–104 (2001).
2. Craig, N. L. The Mechanism of Conservative Site-specific Recombination. 77–105 (1988).
3. Herron, P. R. Mobile DNA II. Heredity 92, 476–476 (2004).
4. Ghosh, K., Guo, F. & Van Duyne, G. D. Synapsis of loxP sites by cre recombinase. J. Biol. Chem. 282, 24004–24016 (2007).
5. Gopaul, D. N., Guo, F. & Van Duyne, G. D. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175–4187 (1998).
6. Guo, F., Gopaul, D. N. & Van Duyne, G. D. Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse. Proc. Natl. Acad. Sci. U. S. A. 96, 7143–7148 (1999).

Keywords: Cre Recombinase, Solution NMR, Dynamics